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Yonago Acta Medica 2017;60:67–70 Short Communication
Corresponding author: Sachiko Nakamoto, PhD naka827@med.tottori-u.ac.jp
Received 2016 December 2 Accepted 2017 January 18
Abbreviations: A. xylosoxidans, Achromobacter xylosoxidans; AG, aminoglycoside; AMK, amikacin; AZT, aztreonam; CFPM, cefepime; CPFX, ciprofloxacin; GM, gentamicin; HIVA, Heart Infusion broth including Vancomycin and Aztreonam; IPM, imi-penem; KM, kanamycin; MIC, minimal inhibition concentration; NEO, neomycin; NET, netilmicin; PIPC, piperacillin; P. aerugi-nosa, Pseudomonas aeruginosa; SM, streptomycin; SPCT, spec-tinomycin; TOB, tobramycin; X-MacVA, Xylose-Macconkey agar including Vancomycin and Aztreonam
Environmental Distribution and Drug Susceptibility of Achromobacter
Xylosoxidans Isolated from Outdoor and Indoor Environments
Sachiko Nakamoto, Misaki Sakamoto, Kana Sugimura, Yuki Honmura, Yuki Yamamoto, Natsumi Goda, Hiroo Tamaki and Naoto Burioka
Department of Pathobiological Science and Technology, School of Health Science, Tottori University Faculty of Medicine, Yonago 683-8503, Japan
ABSTRACT
Achromobacter xylosoxidans is an environmental bacte-rium with multi-drug resistance. We isolated Achromo-bacter xylosoxidans and investigated its susceptibility to 13 drugs. Seventy-eight water samples were collected from rivers and ponds, and 11 samples were swabbed from residential sinks and baths. Nine strains of Achro-mobacter xylosoxidans were isolated from the 89 sam-ples. Five strains, including 2 that were sampled from residential homes, showed high resistance to multiple aminoglycosides. This indicated that Achromobacter xy-losoxidans is widely distributed in various outdoor and indoor environments. Moreover, since these highly resis-tant bacteria were present in indoor environments, cau-tion should be taken for elderly people living at home. Furthermore, a careful assessment should be made for diagnosing and treating compromised hosts.
Key words Achromobacter xylosoxidans; environ-mental distribution; environenviron-mental resident bacterium; multi-drug resistance; opportunistic infection
Achromobacter xylosoxidans (A. xylosoxidans) is a cata-lase-positive, oxidase-positive, and glucose-non-ferment-ing Gram-negative rod. Non-fermentglucose-non-ferment-ing Gram-negative rods have similar properties and are difficult to differ-entiate.1 Furthermore, since A. xylosoxidans is an
envi-ronmental pathogen, it is usually overlooked in routine clinical examinations. A. xylosoxidans is an opportunis-tic bacterium with multi-drug resistance and infections are known to cause clinical difficulties since it becomes
chronic and refractory.2
We previously reported a rare case whereby a strain classified as multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) was further identified as drug-susceptible P. aeruginosa and multidrug-resistant A. xylosoxidans.3 Based on this experience, we became
aware of the importance of surveying the environmental distribution and drug-susceptibility of A. xylosoxidans existing in nature. We devised an isolation procedure suited for the properties of A. xylosoxidans, isolated it from various environmental samples, and investigated the environmental distribution and drug susceptibility of the isolates. The results suggest that more attention to this species is necessary.
MATERIALS AND METHODS Culture media
Heart Infusion broth including Vancomycin and Aztreo-nam (HIVA) broth was prepared by adding 32 μg/mL of vancomycin and aztreonam to heart infusion broth and was used as an enrichment medium. Xylose-Macconkey agar including Vancomycin and Aztreonam (X-MacVA) agar plate was prepared by adding 1% xylose (X) and 20 μg/mL of vancomycin and aztreonam to MacConkey agar and was used as an isolation medium.2
Method for isolation from the environment
Samples were collected from rivers, ponds, irrigation ditches, and residential homes within an area of 20 km of the Yumigahama Peninsula in the west area of Tottori Prefecture, Japan. The samples were either collected in volumes of 15 mL from outdoor water sources or ob-tained from drains of residential sinks and baths using sterilized swabs, which was subsequently suspended with 15 mL of distilled water. These samples were cen-trifuged at 4,000 × g for 15 minutes, and the sediment was cultured in HIVA broth 72 h at 35 °C. Cultured bacterial fluid was cultured on X-MacVA agar 72 h at 35 °C. The colonies that formed on the agar were typed, and the number of colony types was regarded as the number of isolated colonies. A typical colony in each colony type was subjected to differentiation tests. Of the bacteria determined to be Gram-negative rods, only
68 S. Nakamoto et al.
Table 1. The state of isolation of Achromobacter xylosoxidans from environmental materials and the MICs of 13 antibacterial drugs against environmentally isolated Achromobacter xylosoxidans
Samples n
Number of colonies Antibiotics MIC (µg/mL) AGs Isolated
n Differen-tiatedn Identifi edn Identifi edname PIPC CFPM AZT IMP CPFXAMK GM SPCT SM KM TOB NET NEO Water 78 57 45 6 W1 0.5 8 128 1 1 8 4 128 ≥ 128 8 4 4 16 W2 0.25 8 128 1 2 128 32 128 16 128 8 4 128 W3 0.5 4 128 1 1 4 2 ≥ 128 64 8 2 2 8 W4 1 32 64 4 2 ≥ 128 ≥ 128 ≥ 128 ≥ 128 128 64 128 ≥ 128 W5 0.25 8 128 1 2 4 2 128 32 8 2 2 8 W6 16 32 128 1 4 64 64 ≥ 128 ≥ 128 128 16 32 128 Swabs 11 8 8 3 S1 0.5 32 128 2 8 ≥ 128 128 ≥ 128 ≥ 128 ≥ 128 64 128 ≥ 128 S2 0.5 32 64 2 2 ≥ 128 ≥ 128 ≥ 128 128 64 64 128 ≥ 128 S3 0.25 8 128 1 1 4 2 128 64 8 2 4 8 Achromobacter xylosoxidans 600S 0.5 128 128 1 16 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 Samples: water; rivers, ponds, irrigation ditches, swabs; the drains of residential sinks and baths.
Drugs:AG; aminoglycoside; AMK, amikacin; AZT, aztreonam; CFPM, cefepime; CPFX, ciprofl oxacin; GM, gentamicin; IMP, imipen-em; KM, kanamycin; NEO, neomycin; NET, netilmicin; PIPC, piperacillin; SPCT, spectinomycin; SM, streptomycin; TOB, tobramycin. MIC, minimal inhibition concentration.
Fig. 1. Map of the sampling points and A. xylosoxidans isolation. Map of the Yumi-gahama Peninsula extending from Yonago City to Sakaim-inato City, Tottori Prefecture, Japan. The sampling points of A. xylosoxidans-positive and -negative materials are indicat-ed as and , respectively. Sample numbers are shown in the symbols. A. xylosoxidans, Achromobacter xylosoxidans. 30 82 81 80 79 78 77 7 43 45 57 56 4 3 1 14 58 18 19 10 51 49 53 87 59 20 55 15 73 21 89 2 9 8 13 6 5 34 71 65 61 34 41 16 11 12 40 17 15 39 36 37 35 38 60 54 52 50 46 44 42 74 88 72 66 62 86 85 84 76 75 68 67 70 69 64 63 83 33 32 31 29 28 27 26 25 24 23 22 12 1 58 583465 65 65 65 3465 3465 65 11 40 39 39 40 39 40 40 39 40 40 38 40 40 39 40 38 40 39 40 40 39 40 38 40 39 40 38 38 88 65 66 65 65 66 65 65 66 65 34 65 34 66 34 65 34 34 65 34 66 34 65 34 65 66 65 66 66 38 6238 62 62646666 63 6366 6366 6463 6466 64666366 64666363 14 14 202020 1919 63 63 31 22 22 666363 84 84 6785858385696967686867686767686768676768 84858385 8483858327268327832783838383262626262323232423242324226322632222222222 32 32 31 272523232222 42 42 42 43 50444444 45 45 45 464545 4645 46 46 4645455043505050504949434343 4645 46 465050435043504350445043504350435043504350504350445044494349434949494349494349434943494349434944434444434343444344434344434344434344 51 49 Yonago city Sakaiminato city Hiezu son 5 km those that were positive for catalase and oxidase tests
were further tested for their non-fermentability, growth ability in stab culture, citrate utilization ability, and nitrate-reducing ability. These differential test-positive bacteria were identified using ID test NF-18 (Nissui Pharmaceutical, Tokyo, Japan), a simple identification kit for glucose-non-fermenting Gram-negative rods. A fi nal identifi cation was made according to a 16S rDNA base sequence.4
Bacterial strains
Nine environmentally isolated A. xylosoxidans and A. xylosoxidans 600S strains3 were used.
Drug susceptibility tests
The minimal inhibition concentration (MIC) was de-termined by the agar plate dilution method using the Clinical and Laboratory Standard Institute procedure. The following drugs were tested: piperacillin (PIPC), cefepime (CFPM), aztreonam (AZT), imipenem (IPM), ciprofloxacin (CPFX), amikacin (AMK), gentamicin (GM), streptomycin (SM), kanamycin (KM), netilmicin (NET), spectinomycin (SPCT), neomycin (NEO) and tobramycin (TOB).
RESULTS
Table 1 shows the isolation state of A. xylosoxidans from various samples and the MICs of 13 antibacterial drugs
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Distribution and drug susceptibility of A. xylosoxidans for the isolated A. xylosoxidans. Figure 1 shows the map
of the sampling points and A. xylosoxidans isolation. State of isolation
Of the 89 samples, 9 strains of A. xylosoxidans were identified. The identification rate was 10.1% (6 strains (7.7%) in outdoor water samples and 3 strains (27.3%) in swab samples from residential homes) (Table 1). The species was identified in a wide sampling area (Fig. 1). Evaluation of the isolation and identification meth-ods
The number of isolated colonies that formed on X-Mac-VA agar was low, at around 70% per sample. The differ-entiation tests showed that 81.5% of the isolated colonies were positive for oxidase and catalase. Based on the bacterial identification tests, 10.1% of the colonies were identified as A. xylosoxidans. Furthermore, according to the isolation and identification protocol, of the red col-onies that formed on X-MacVA agar, the large colcol-onies were identified as Pseudomonas fluorescens and the pin-point colonies as A. xylosoxidans. Finally, A. xylosoxi-dans was confirmed using the 16 rDNA base sequence. Drug susceptibility tests
The strains were susceptible to β-lactams (PIPC, IMP, CFPM) except for monobactam (AZT), and new quino-lone (CPFX). Of the 8 aminoglycosides (AGs; AMK, GM, SPCT, SM, KM, TOB, NET, NEO) that were tested, the MIC of SPCT was 128~ ≥ 128 μg/mL for all strains. However, drugs other than SPCT and SM had low MICs for 4 strains (W2, W3, W5, S3) and high MICs for 5 strains (W1, W4, W6, S1, S2), which closely resembled the resistance pattern of the A. xylosoxidans 600S strain. Of the 5 multidrug-resistant strains (W2, W4, W6, S1, S2) in all, 2 were isolated from residential homes (S1, S2) (Table 1).
DISCUSSION
Since A. xylosoxidans is an environmental bacterium, it is rarely detected in routine clinical examinations, and, if it is detected, its differentiation and identification is difficult as it is often mistaken for P. aeruginosa. A. xy-losoxidans has multidrug resistance and erroneous iden-tification of the bacteria will lead to inappropriate se-lection of drugs. Therefore, information concerning the distribution of this species and the drug susceptibility of bacteria distributed in the environment is important.
The enrichment and isolation media used in this study, which included AZT resistance and xylose de-gradability, showed high selectivity. Red colonies that formed on X-MacVA agar were likely
glucose-non-fer-menting Gram-negative Pseudomonas and A. xylosoxi-dans. Pin-point colonies, in particular, were considered to be an important criterion for detecting A. xylosox-idans.2 Although the differential tests are effective for
confirming major properties of bacteria, they are not able to precisely determine the species. Therefore, a definitive diagnosis based on the 16S rDNA base sequence is im-portant. Since Pseudomonas and A. xylosoxidans have similar properties, they are often misidentified or over-looked due to differences in their growth ability.3 Proper
knowledge regarding the properties of A. xylosoxidans is necessary to avoid misdiagnoses in routine examina-tions.
Amoureux et al.2 isolated A. xylosoxidans in a wide
area and reported identification rates of 28% and 12% from samples collected from the outdoor and indoor en-vironments, respectively. In our study, the identification rate for samples collected from indoor residences was high at approximately 30% for bath drains. In contrast, the identification rate was relatively low for outdoor wa-ter sources, which may have been due to the small sam-ple volume of 15 mL. In addition, A. xylosoxidans has a biofilm-forming ability.5 Biofilm can form in the drains
of sinks and baths, which may have led to the high bac-terial concentrations in the swab samples. Considering these points, we deem our bacterial isolation procedure appropriate. Furthermore, the wide distribution of this species in indoor environments was demonstrated.
The 9 strains of A. xylosoxidans isolated from the various environments resembled the common multi-drug resistance pattern,6 and some drugs showed for
high MICs, AGs in particular. The MICs of the 8 AGs for the A. xylosoxidans 600S strain 3 were ≥ 128 μg/
mL, and 5 (W2, W4, W6, S1, S2) strains were isolated from both the outdoor and indoor samples. Since A. xylosoxidans can be mistaken for P. aeruginosa, care is necessary when selecting drugs for Gram-negative rod infections. In addition, this species is drug-resistant and is distributed in wet areas. The latter feature may cause problems since the bacteria may accumulate in the sinks of residential kitchens and baths. Currently, residences of elderly people in need of medical and nursing care are increasingly transitioning from medical facilities to residential homes, especially care homes for the elderly. Sufficient attention is necessary for vulnerable elderly people and compromised hosts who live at home. The authors declare no conflict of interest.
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REFERENCES
1 Yabuuchi E, Ohyama A. Achromobacter xylosoxidans n. sp. from human ear discharge. Jpn J Microbiol. 1971;15:477-81. PMID: 5316576.
2 Amoureux L, Bador J, Fardeheb S, Mabille C, Couchot C, Massip C, et al. Detection of Achromobacter xylosoxisidans in hospital, domestic, and outdoor environmental samples and comparison with human clinical isolates. Appl Environ Mi-crobiol. 2013;79:7142-9. PMID: 24038696.
3 Nakamoto S, Goda N, Hayabuchi T, Tamaki H, Ishida A, Suzuki A, et al. Properties of Achromobacter xylosoxidans highly resistant to aminoglycoside antibiotics. Jpn J Antibiot. 2016;69:113-8. PMID: 27544979.
4 Liu L, Coenye T, Burns JL, Whitby PW, Stull TL, LiPumal
JJ. Ribosomal DNA-directed PCR for identification of Ach-romobacter (Alcaligenes) xylosoxidans recovered from spu-tum samples from Cystic Fibrosis patients. J Clin Microbiol. 2002;40:1210-3. PMID: 11923333.
5 Jakobsen TH, Hansen MA, Jensen PØ , Hansen L, Riber L, Cockburn A, et al. Complete genome sequence of the Cystic Fibrosis pathogen Achromobacter xylosoxidans NH44784-1996 complies with important pathogenic phenotypes. PloS One. 2013;8:1-17. PMID: 23894309.
6 Glupczynski Y, Hansen W, Freney J, Yourassowsky E. In vitro susceptibility of Alcaligenes denitrificans subsp. xylosoxidans to 24 antimicrobial agents. Antimicrob Agents Chemother. 1988;32:276-8. PMID: 3163242.
